Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of acquired immune deficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including the long terminal repeats (LTRs), the gag, pol, and env genes, and other genes. This retrovirus carries a number of viral antigens which are potential candidates either alone or in concert as vaccinal agents capable of inducing a protective immune response.
Among the more promising of the HIV-1 antigens is the viral envelope glycoprotein (gp160) or specific fragments thereof. The env gene encodes the 160 kilodalton (kd) precursor glycoprotein of the viral envelope. gp160 is cleaved posttranslationally into a 120 kd glycoprotein (gp120) and a 41 kd glycoprotein (gp41), which are present at the virus surface.
These viral glycoproteins assume a tertiary structure as viral spikes protruding outwards from the surface of the viral particle. About 70 to 80 spikes are believed to be associated with each newly synthesized viral particle. As the viral particle ages, the spikes disappear, apparently because the association between the gp120 and gp41 is weak. Thus, for newly synthesized viral particles, this viral glycoprotein spike is believed to be the most immediate target accessible to the immune system following infection.
Virus neutralizing antibodies have been reported directed against gp120 and gp41 epitopes. It has been specifically noted that a target site for type specific neutralizing antibodies is located in the 3' half of the gp120 glycoprotein molecule.
The env gene of HIV-1 has thus been the target of numerous recent investigations. Expression of glycosylated gp160 has previously been obtained in mammalian cells and certain baculovirus insect cells by groups which have also reported the induction of both humoral and cellular immune responses to these antigens. gp120 has been expressed recombinantly with the use of heterologous promoters in several systems. See, e.g., S. Chakrabarti et al, Nature (London), 320: 535 (1986); S. I. Hu et al, Nature (London), 320: 537 (1986); and M. P. Kieny et al, Biotechnology, 4: 790 (1986).
L. A. Lasky et al, Science, 233: 209-212 (1986) constructed a number of plasmids containing mutant env genes for tranfection into mammalian cells, specifically Chinese hamster ovary (CHO) cells. These researchers secreted a gene product encoded in a plasmid containing the first 50 amino acids of the glycoprotein D (gD) protein joined in phase to an amino acid sequence (#61-#531) of the env protein, an HBsAg polyA signal, a DHFR gene and the SV40 origin of replication. A recombinant envelope antigen was produced containing 25 amino acids of gD at its amino terminus and lacking 30 residues from the mature processed from of gp120, and also having a deletion of the gp41 sequence (about 20 amino acids of the carboxyl terminus to the actual 160 kd precursor processing site). The resulting gene was 520 amino acids in length. When transfected into CHO cells, the cell-conditioned supernatants contained a 130 kd protein, called gp130.
Fox, Biotechnology, 6: 116 (1988) reports the VAXSYN HIV-1 vaccine developed by MicroGeneSys. This report does not disclose any details of this vaccine.
D. L. Lynn, et al, in "Mechanisms of Control of Gene Expression", Eds. Allan R. Liss Inc., pp. 359-368 (1988) disclose the cloning of the entire gp160 gene behind the polyhedron promoter of the baculovirus Autographacalifornica. These insect cells infected with the recombinant virus express a protein that is released from the cell upon lysis. This protein co-migrates with gp160, is not cleaved into gp120 and gp41, and is glycosylated and associated with the cell membrane. When deglycosylated with N-glycanase, the protein had a molecular weight of approximately 96 kd. The recombinant protein was immunoreactive with protein from HIV-infected H9 cells, with antisera to a recombinant fraction of gp120, with gp120 itself, with a peptide fragment of gp41, and with human AIDS sera.
tPA can also be prepared by recombinant DNA techniques. Isolation of mRNA for tPA is disclosed, e.g., by Opdenakker et al., Eur. J. Biochem. 12:269 (1982). Isolation of cDNA for part of tPA is disclosed by Edlund et al., Proc. Natl. Acad. Sci. USA 80:349 (1983). Cloning of cDNA for tPA in E. coli is reported by Pennica et al., Nature 301:214 (1983). Cloning of cDNA for tPA in E. coli and in Chinese Hamster Ovary cells by application of routine recombinant DNA procedures is disclosed by Goeddel et al., EP-A-93,619 and by Levinson et al., EP-A-117,059. Goldberg et al., PCT patent application WO85-03949, disclose expression of tPA in E. coli. Meyhack et al., EP-A-143,081, disclose expression of tPA in yeast. Robinson, WO84-01786, discloses a modified tPA lacking all or a portion of the carbohydrate moieties present in native tPA.
The development of Drosophila cell cultures which are stable and can be grown under laboratory conditions have been reported. See Schneider, J. Embryol. Exp. Morphol. 27:353 (1972). Various vectors systems containing specific coding sequences have been inserted into Drosophila under the control of the Drosophila heat shock promoter or COPIA promoters. DiNocera et al., Proc. Natl. Acad. Sci. USA 80:7095 (1983). More recently, mRNA encoding the heat-shock promoters has been translated in Drosophila cells at high rates, McGarry et al., Cell 42:903 (1985).
B. J. Bond et al, Mol. Cell. Biol., 6(6): 2080 (1986) disclose the structure of the Drosophila melanogaster actin 5C gene. The report discusses the two transcription start sites of the actin 5C gene and fusions between the promoter sequences and bacterial chloramphenicol acetyltransferase gene inserted into D. melanogaster host cells.
H. Johansen et al, 28th Annual Drosophila Conference, p. 41 (1987) is an abstract by the inventors of the present application which briefly states that E. coli gal K genes regulated by a Drosophila metallothionein promoter were expressed in Drosophila cell lines.
A. Vanderstraten et al, Proceedings of the 7th International Conference on Invertebrate and Fish Tissue Culture, Abstract, University of Tokyo Press, Japan, (1987) and A. Vanderstraten et al, in "Invertebrate and Fish Tissue Culture", Eds. Y. Kuroda et al, Japan Scientific Societies Press, Tokyo, pp. 131-134, (1988) are also publications by the present inventors which discuss a hygromycin B selection system.
It is thus an object of this invention to introduce, select and overexpress heterologous genes in a Drosophila expression system.